Citation for published version (APA): Wientjes, K. J. C. (2000). Development of a glucose sensor for diabetic patients Groningen: s.n.

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1 University of Groningen Development of a glucose sensor for diabetic patients Wientjes, Klaas Jan Cornelis IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2000 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Wientjes, K. J. C. (2000). Development of a glucose sensor for diabetic patients Groningen: s.n. Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date:

2 Summary This thesis describes the development of a glucose measurement system designed to monitor the glucose concentration of diabetic patients continuously. Diabetes Mellitus is a disorder of metabolism of carbohydrate, fat and protein associated with a relative or absolute insufficiency of insulin secretion and with various degrees of insulin resistance. The World Health Organisation (who) has estimated that diabetes affects about 135 million people worldwide. The two most occurring types are type-1 and type-2 diabetes mellitus. They both account for 99% of all prevalence of diabetes. In untreated state the disease is characterised by hyperglycaemia (blood glucose concentrations higher than 10 mm). It is very important to keep blood glucose concentrations within a narrow range of 3 to 10 mm. Glucose concentrations under 3 mm (hypoglycaemia) impair brain function, whereas glucose concentrations higher than 10 mm exceed the renal glucose reabsorption threshold, which results in wasting of glucose. In addition, protracted hyperglycaemia causes degenerative complications in the long-term [2]. For type-2 diabetic patients a diet, oral hypoglycaemic agents and/or the administration of insulin usually mange to regulate the blood glucose concentration. First priority is to reduce chronic hyperglycaemia and the associated long-term degenerative complications. Type-1 diabetic patients have an absolute insulin deficiency and can only be treated by insulin injections. Main objective is to normalise the blood glucose concentration in order to reduce long-term complications. Hypoglycaemia, on the other hand, is the most common side effect of treatment with insulin and represent the immediate subject of concern for the patient. The development of self-monitoring of blood glucose (finger-prick method) has been a major improvement in the insulin therapy. However, the number of measurements done with the finger-prick method is limited and will only provide infor- 149

3 mation about blood glucose values at intermittent moments. Continuous in vivo glucose monitoring may therefore be an improvement and may contribute to a more adequate insulin administration. In addition, a glucose sensor could be of use in the early detection of hypoglycaemia. In chapter 1 some basic concepts like classification, causes, complications and treatment of diabetes mellitus are discussed. Type-1 diabetes is a result of a chronic autoimmune destruction of the pancreatic beta cells resulting in an absolute insulin deficiency. In type-2 diabetic patients organs are less sensitive for the action of insulin and/or the production of the beta cells is insufficient. Type-2 diabetic patients can be treated with a diet, hypoglycaemic agents and/or insulin injections whereas type-1 diabetic patients can only be treated with insulin injections. Especially for type-1 diabetic patients, insulin injections in combination with self-monitoring of blood glucose have improved diabetic control. However, it is still difficult to achieve near normoglycaemia. Two other approaches to improve metabolic control in type-1 diabetics are discussed in this chapter: 1. Transplantation of the pancreas or isolated islets of Langerhans. 2. The use of continuous glucose monitoring systems preferentially combined with a feedback controlled insulin dosage system. Although transplantation of especially the pancreas in combination with the kidney has a success rate of approximately 75% it remains to be seen whether the majority of type-1 diabetics will profit. This, because of the shortage of suitable donors and the lifetime use of immunosuppression agents. Continuous glucose measurements may be an improvement over self-monitoring using the finger prick method. Self-monitoring of blood glucose suffers from the fact that it is discontinuous. A continuous glucose monitoring system would therefore provide an alternative to the present discrete methods of glucose monitoring. Based on the sensor signal the patient can anticipate and thus regulate his blood glucose concentration. A technical advanced application is an insulin delivery system that is feedback controlled by a continuous glucose measurement system (artificial beta cell). In this case, insulin administration is based on the on-line measurement of the glucose sensor without or with minimal interference of the patient. Possible 150

4 methods of glucose monitoring, as well as in vivo applications and requirements of glucose sensors are discussed in this chapter. In chapter 2 various minimally invasive glucose sensors and their basic detection principles are reviewed. There are different types of glucose sensors developed depending on the used detection- and glucose extraction techniques. However, the majority of sensors monitor the enzyme-catalysed oxidation of glucose. Glucose is oxidised to gluconic acid by the enzyme glucose oxidase (god) under consumption of oxygen and producing of hydrogen peroxide. Electrodes based on amperometric or potentiometricoperating principles can monitor this reaction i.e. the depletion of oxygen, or the amount of hydrogen peroxide/gluconic acid produced. Principles and methods of these enzyme based glucose sensors and their subsequent benefits/shortcomings are discussed. Hydrogen peroxide glucose sensors are the most used because of their relative ease of manufacturing and the possibility of constructing them in small sizes. Special attention should be paid to the safety of these sensors when they are used for in vivo measurements. In most cases a needle-type glucose is inserted directly into the tissue of the subject. With these needle-type sensors, god is immobilised on an electrode. Small (bio-)molecules may foul the electrode space, causing rapid loss of sensitivity and in addition, produced hydrogen peroxide deactivates the enzyme god. Hence the poor in vivo performance of nearly all the hydrogen peroxide based glucose sensors. An alternative is the oxygen-based glucose sensor where the electrode is protected by a hydrophobic membrane that is only permeable for gases and not for biomolecules. In addition, the enzyme catalase is co-immobilised with god to forestall peroxide-mediated deactivation of this god. Unfortunately, up till now, oxygen-based glucose sensors can not be miniaturised to the same extent as peroxide-based sensor. Also a disadvantage is the dependency on a constant environmental oxygen concentration in the tissue surrounding the sensor. Because these disadvantages, of both the hydrogen peroxide based glucose sensor as well as the oxygen electrode based glucose sensor, a number of other sensor concepts have been explored which are also discussed in chapter 2. One of these concepts is the use of microdialysis as a special means of transport to deliver glucose from the body to the glucose sensor. Basically the technique comprises 151

5 the implementation in tissue or blood compartment of a small hollow semipermeable dialysis-membrane construction (named probe) that is perfused with a water-based solution (termed perfusate). Glucose diffuses from the tissue or blood into the perfusate and is transported to the glucose measurement system. The technique circumvents various disadvantages of the direct implanted glucose sensors and seems a promising approach for continuous subcutaneous glucose measurement. Microdialysis based glucose sensors are discussed in greater detail in chapter 2 and through the remainder of this thesis. The effect of sensor or microdialysis probe implementation on glucose measurement is still partially unclear and is also briefly discussed in this chapter. Chapter 3describes the development a glucose measurement system based on the work of Schoonen and Schmidt [1]. A major disadvantage of the system of Schoonen and Schmidt is that a god/catalase solution is used as perfusion fluid. This means that there is a potential danger of enzyme leakage through the dialysis tube. This makes the clinical use of such a system not appropriate. We redesigned the closed-flow microdialysis perfusion system of the sensor to minimise the risk of enzyme leakage without losing the benefits of a closed perfusion system. Two new flow system designs are described in this chapter: 1. The dual circulation system. 2. The single circulation system. The dual circulation system combines, as it names indicates, two flow systems. A saline solution is used to perfuse the microdialysis probe. After the probe outlet, saline is mixed with a god/catalase solution. Now, god reacts with the glucose present under the consumption of oxygen. An oxygen electrode measures this diminution of dissolved oxygen in the mix. The amount of diminution is a measure for the amount of glucose present. Ultrafiltration is used to separate the two types of flow. For reasons of safety, a filter containing active carbon was inserted in the saline circulation to adsorb and block enzyme molecules that possibly leaked through the ultrafiltration membranes. Validation experiments of the these carbon filters are described. No god molecules could be demonstrated in the perfusion fluid 152

6 after a carbon filter. To further decrease the risk of enzyme leakage the singular circulation flow system was developed. In this system the god/catalase solution was retained in an enzyme reactor. Isotonic saline was used to perfuse the microdialysis probe. To prevent the accumulation of glucose within the system a glucose eliminator was developed. A detailed construction of both systems is given in this chapter. Both advantages and disadvantages of the two systems are discussed. For in vivo studies and further development, the singular circulation system is most suitable. This, because the single circulation system has the lowest risk of enzyme leakage, lowest energy consumption and the most promising possibilities for further miniaturisation. Chapter 4 describes various in vitro characteristics of the singular circulation system based glucose measurement system (sc-gms). We determined the accuracy of the sensor measurements, the sensor sensitivity for glucose, the sensor response times, the influence of temperature on the sensor measurements and the overall stability. The results show that it is possible to measure glucose reliably with the new designed flow system. The in vitro drift of the total system is acceptable to measure for a period of at least 2 weeks. In Chapter 5, continuous in vivo measurements with the glucose measurement system are presented. The feasibility of the system was tested in an ambulatory study with ten healthy volunteers with a general objective to measure reliably up to seven days. Additional effects on the sensor measurement of temperature changes and exercise of the subject were also investigated. Before application in vivo, the sensor was calibrated in vitro with known glucose solutions. Also the temperature dependence for each system was assessed to correct for temperature changes. On day one the subjects arrived fasting at the hospital where an oral glucose tolerance test (oggt) was performed. The purpose of this test was to investigate the magnitude of the lag-time between a change in blood glucose and the sensor signal. Additional tests were performed in the hospital to assess the effect of temperature and physical exertion on the sensor measurements. On day 2 to the end of the experiment, subjects checked in every morning in the hospital to download the measurement data and to examine the functioning of the sensor. 153

7 The study showed that it was possible to monitor the glucose concentration ambulatory with the sensor for at least two to three days. Biggest problem was the lack of robustness of the system. This resulted under daily life conditions in obstructions and leakage of the perfusion flow system. The correlation between blood glucose concentration and sensor measurements is well within clinical accuracy. A maximum lag-time was found of 10.5 minutes in the ascending part of the oggt curve, whereas a maximum lag-time of 9.5 minutes was found in the descending part. An interesting finding during these studies was that the recovery of the microdialysis probe was increasing in time. The recovery of blood capillaries and the subsequent increase of glucose supply may be an explanation of this phenomenon (see also chapter 7). Chapter 6 deals with the qualitative and quantitative aspects of microdialysis. This because microdialysis is the base technique used in the glucose measurement system and for a proper interpretation of measured data a solid understanding of these processes are helpful. In this chapter an attempt is made to answer the question to what extent in vitro calibrations can be used for in vivo measurements. Mathematical models of Jacobson [245] and Bungay [257] are shortly discussed. Using in vitro data, in vivo data and a mathematical model of the probe, the in vivo mass transfer coefficient of glucose could be calculated. The results of this calculation show that the estimated tissue resistance is a factor three lower than the membrane resistance. This means that the depletion of glucose from the tissue around the probe is not a limiting factor to the glucose recovery in the flow range used in the experiments ( µl/min). Chapter 7 describes glucose measurements in subcutaneous adipose tissue by microdialysis for 3 weeks in five healthy subjects. We evaluated the change in recovery in time. In these experiments we tried to derive the true interstitial glucose concentration in adipose tissue (or in other words: what is a microdialysis probe really measuring?). The kinetics of glucose transport in adipose tissue was also investigated. We used perfusion flow rates from 0.5 µl/min to 20 µl/min for microdialysis probes with a membrane length between 10 and 35 mm and perfused various glucose solutions as introduced by Lönnroth et al. [221]. In each subject four probes were 154

8 inserted, two on each side of the umbilicus. Each day, an experiment was performed either with eight different flow rates or an experiment with various glucose concentrations. Using vials, dialysate was sampled twice for each flow rate or each perfused glucose concentration. The glucose content in the collected dialysate was estimated spectrophotometrically. At the beginning and at the end of the sampling period, a blood glucose measurement was taken. The study design and a detailed description of methods used are given in this chapter. The results showed that the initial glucose recoveries increased for 6-9 days until a plateau was reached. Especially the (individual) change in recovery during the 1st day was significant. Probably insertion of the probe causes some trauma to cells and capillaries, creating a not fully functional layer of tissue around the probe that may present a barrier to diffusional glucose transport. The repair of the tissue microstructure around the probe may well take a rather long time and is accompanied by a steady increase of the glucose recovery. At a flow rate of zero, the equilibrium concentration is not effected by any kinetic parameter (Lönnroth method). The concentration equals the actual concentration in the surrounding tissue and depends solely on the balance between the supply of glucose by the arteries and the uptake of glucose by cells. Our results show equal glucose values for capillary blood and the equilibrium glucose concentration when no implantation effects are present. We think therefore that not some universal interstitial concentration is the driving force for diffusion to the probe but that it is the capillary blood glucose concentration itself. This means that for microdialysis experiments in adipose tissue in general, for substances not produced in the tissue around the probe, capillary blood is a better reference than venous plasma. Based on this histological picture, adipose tissue is better described by a tissue matrix of cells and capillaries in which the transport of glucose in tissue is governed by convective flow of blood inside the vascular bed and not so much by diffusion in the interstitial space. This matrix model views transport of glucose as a twostage process. First, convective flow in the vascular bed for transport of glucose to the vicinity of the probe. Second, diffusional flow across the capillary wall, a small layer of interstitial fluid and the membrane wall of the probe. The matrix model integrates diffusional transport of glucose across the capillary wall, the interstitial fluid layer and the probe membrane. The observed 155

9 implementation effect on the recovery may well impede a short-term application of continuous microdialysis in adipose tissue. Only when the recovery is stabilised, dialysate glucose concentrations correlate directly with the blood glucose concentration. We therefore recommend that for reliable continuous glucose measurement one should wait until recovery has stabilised. 156